Acute myeloid leukemia (AML), a highly lethal subtype of leukemia, has a 5-year survival rate of <20%. With the advent of next-generation sequencing, a number of specific genetic lesions that drive AML and provide prognostic information have been identified. Previous work by our group (co-PD/PI, Druker) provided proof that imatinib, a targeted ABL kinase inhibitor that blocks activity of the BCR-ABL fusion protein in chronic myeloid leukemia (CML), dramatically improves patient survival. Similar approaches have been applied to AML, a more genetically complex leukemia, and some drugs have improved outcomes, but none have been as successful as ABL inhibitors for CML due to incomplete responses and the rapid development of resistance. Project 1 of this DRSC Program, Drug Combinations to Circumvent Resistance (D2CR), will focus on understanding intrinsic mechanisms of enhanced drug sensitivity or resistance, with the goal of devising novel therapeutic strategies. For this Project, our long-term goals are to nominate drugs that enhance upfront drug sensitivity and/or circumvent resistance for use in combination strategies that will be tested in clinical trials. Our immediate goals are to identify essential target genes and pathways contributing to sensitivity or resistance to specific drugs and to validate their roles using cell lines, patient samples, and xenograft-derived cells. These goals are based on our central hypothesis that the heterogeneous genetic landscape of AML, in tandem with complex signaling feedback loops, contributes to intrinsic mechanisms of drug sensitivity and resistance. Project 1 will provide critical preclinical data to advance drug candidates for use in combinations tested in primary patient samples and xenograft models in Project 3. To accomplish these goals, 3 Aims are proposed: 1) Identify genetic mechanisms of drug sensitivity/resistance in AML cell lines through essential-gene and drug-resistance screens ? We will perform genome-wide CRISPR/Cas essential gene and re-sensitization screens to identify gene targets and pathways contributing to enhanced drug activity or resistance. Data generated by the combination of these 2 screens will provide key insights into cell-intrinsic mechanisms of sensitivity or resistance to 5 select drugs (crenolanib, quizartinib, ruxolitinib, trametinib, and venetoclax) in AML cells with diverse genetic backgrounds. 2) Computationally validate, refine, and inform candidate pathways and genes contributing to intrinsic mechanisms of drug sensitivity or resistance ? This iterative modeling step will leverage the intrinsic genetic factors identified by our in-house Cancer Targetome Knowledgebase and will prioritize targets for further validation in Aim 3. 3) Validate new gene targets in hypothesis-driven, focused CRISPR/Cas experiments ? We will develop a targeted CRISPR/Cas sgRNA library to perturb genes hypothesized to contribute to drug sensitivity or resistance in cell line models, xenografts, and primary patient samples. We expect to identify gene targets for further interrogation in drug combination testing in Project 3.